A family of proteins found on the surface of cells is known as the ATP-binding cassette (ABC) family of transmembrane reporter proteins. Expression of these proteins affects the transport of drugs into cells. This family of proteins includes the trans-membrane ATP-dependent drug translocation protein P-glycoprotein (Pgp; Nooter, K. and Sonneveld, P. Leuk. Res. 1993 18:233-243; Biedler, J. L. Cancer Res. 1994 54:666-678; Kerbel et al. Cold Spring Harbor Symp. Quant. Biol. 1994 59:661-672; Broxterman et al. Curr. Opin. Oncol. 1995 7:532-540; and List, A. F. Leukemia 1996 10:937-942); the Multi-drug Resistance-associated Protein (MRP) whose overexpression is associated with multi-drug resistance (Demolombe, S. and Escande, D. TIPS 1996 17:273-275); the Cystic fibrosis Transmembrane Conductance Regulator protein (CFTR), mutations of which cause cystic fibrosis (Schneider et al. British J. Cancer 1989 60:815-818); and the Sulfonourea Receptor (SUR) protein (Fojo et al. Proc. Natl. Acad. Sci. USA 1987 84:265-269). The ability to modulate the expression of these proteins has broad application in a variety of clinical situations including multi-drug resistance in cancer and cystic fibrosis.
Pgp is expressed in a variety of normal tissues including liver, kidney and colon and tumors arising from these tissues usually over-express Pgp as part of their multi-drug resistance (MDR) phenotype (Cole et al. Science 1992 258:1650-1654; Roninson, I. B. Biochem. Pharmacol. 1992 43:95-102; Arceci, R. J. Blood 1993 81:2215-2222; and Merkel et al. J. Clin. Oncol. 1989 7:1129-1136). However, Pgp can also be over-expressed in tumors from tissues that do not normally express this protein, such as breast and ovarian tissues (Arceci, R. J. Blood 1993 81:2215-2222; and Ihnat et al. Clin. Cancer Res. 1997 3:1339-1346). The mechanism of Pgp up regulation in tumors in vivo is still unclear, but can occur de novo as in acute myologenous leukemia (AML) (Gregorcyk et al. Ann. Surg. Oncol. 1996 3:8-14; Koh et al. Yonsei Medical Journal 1992 33:137-142; Dalton, W. S. and Sikic, B. I. J. NIH Res. 1994 6:54-58; Cole et al. Science 1992 258:1650-1654; Demolombe, S. and Escande, D. TIPS 1996 17:273-275; Schneider et al. British J. Cancer 1989 60:815-818; Fojo et al. Proc. Natl. Acad. Sci. USA 1987 84:265-269; Roninson, I. B. Biochem. Pharmacol. 1992 43:95-102; Arceci, R. J. Blood 1993 81:2215-2222; and Merkel et al. J. Clin. Oncol. 1989 7:1129-1136) or can be acquired over the course of cancer treatment as in breast and ovarian cancer (Merkel et al. J. Clin. Oncol. 1989 7:1129-1136; Ihnat et al. Clin. Cancer Res. 1997 3:1339-1346; Hamilton, J. W. and Wetterhahn, K. E. Mol. Carcinogens. 1989 2:274-286; and McCaffrey et al. Mol. Carcinogens 1994 10:189-198).
MDR1 gene transcription and MDR1 mRNA expression can be induced by certain DNA damaging agents, including chemotherapeutic drugs such doxorubicin, simple alkylating agents such as methyl methanesulfonate, and genotoxic chemical carcinogens that induce bulky DNA adducts such as aflatoxin B1 and 2-acetylaminofluorene. In contrast, Pgp mRNA and overall protein expression has been shown to be significantly suppressed by treatment with DNA crosslinking agents, including the cancer chemotherapy drugs, mitomycin C (MMC), cisplatin, carboplatin, and BMS181174, and the carcinogen, chromium(VI). The principal mechanism for the suppression appears to be a down-regulation of MDR1 gene transcription occurring immediately after drug treatment.
However, in a March 1999 Abstract by Maitra et al., it is suggested that there may be a second point of Pgp regulation that mediates changes in Pgp trafficking from an intracellular pool to the cell surface in direct response to a toxic chemical challenge. This suggestion is based upon experiments with a gene construct expressing a Pgp-green fluorescent protein fusion protein under the control of the MDR1 gene promoter in MDCK. MMC treatment was shown to increase membrane levels of Pgp-GFP 6-18 hours after treatment. These levels were reported to subsequently decrease to half the control value by 40 to 60 hours and then return to normal. No details regarding preparation of this construct are disclosed in the Abstract.
Like Pgp, CFTR is a member of the ABC family of transmembrane reporter proteins. Hundreds of different individual CFTR mutations falling into five functional classes have been identified, including missense mutations, frameshifts, in-frame deletions, and splicing mutants. A single mutation resulting in a deletion of the phenylalanine at position 508 of the CFTR protein, known as ΔF508, accounts for approximately 67% of mutations in all CF patients. This mutation results in improper CFTR protein folding and trafficking such that functional CFTR does not reach the cell membrane surface. Experiments in cell culture that are able to overcome the blockade of this mutant CFTR from reaching the cell surface indicate that if ΔF508 reaches the cell membrane it functions normally.
Gene constructs and cells transfected with these constructs have now been produced that have the ability to affect expression of these ABC reporter proteins.